No abstract

We performed a genome-wide association study for primary open-angle glaucoma (POAG) in 1,007 cases with high-pressure glaucoma (HPG) and 1,009 controls from southern China. We observed genome-wide significant association at multiple SNPs near ABCA1 at 9q31.1 (rs2487032; P = 1.66 × 10(-8)) and suggestive evidence of association in PMM2 at 16p13.2 (rs3785176; P = 3.18 × 10(-6)). We replicated these findings in a set of 525 HPG cases and 912 controls from Singapore and a further set of 1,374 POAG cases and 4,053 controls from China. We observed genome-wide significant association with more than one SNP at the two loci (P = 2.79 × 10(-19) for rs2487032 representing ABCA1 and P = 5.77 × 10(-10) for rs3785176 representing PMM2). Both ABCA1 and PMM2 are expressed in the trabecular meshwork, optic nerve and other ocular tissues. In addition, ABCA1 is highly expressed in the ganglion cell layer of the retina, a finding consistent with it having a role in the development of glaucoma.

Various immune response pathways are altered during early, predegenerative stages of glaucoma; however, whether the early immune responses occur secondarily to or independently of neuronal dysfunction is unclear. To investigate this relationship, we used the Wld s allele, which protects from axon dysfunction. We demonstrate that DBA/2J.Wld s mice develop high intraocular pressure (IOP) but are protected from retinal ganglion cell (RGC) dysfunction and neuroglial changes that otherwise occur early in DBA/2J glaucoma. Despite this, immune pathways are still altered in DBA/2J.Wld s mice. This suggests that immune changes are not secondary to RGC dysfunction or altered neuroglial interactions, but may be directly induced by the increased strain imposed by high IOP. One early immune response following IOP elevation is up-regulation of complement C3 in astrocytes of DBA/2J and DBA/ 2J.Wld s mice. Unexpectedly, because the disruption of other complement components, such as C1Q, is protective in glaucoma, C3 deficiency significantly increased the number of DBA/2J eyes with nerve damage and RGC loss at an early time point after IOP elevation. Transcriptional profiling of C3-deficient cultured astrocytes implicated EGFR signaling as a hub in C3-dependent responses. Treatment with AG1478, an EGFR inhibitor, also significantly increased the number of DBA/2J eyes with glaucoma at the same early time point. These findings suggest that C3 protects from early glaucomatous damage, a process that may involve EGFR signaling and other immune responses in the optic nerve head. Therefore, therapies that target specific components of the complement cascade, rather than global inhibition, may be more applicable for treating human glaucoma.G laucoma is a common disorder characterized by the loss of retinal ganglion cells (RGCs) and degeneration of the optic nerve (1-3). Intraocular pressure (IOP) elevation is a major risk factor for developing glaucoma (4). Harmful IOP leads to changes in immune response pathways in nonneuronal cells, such as astrocytes and microglia/monocytes (5-9). Similar changes occur in many neurodegenerative diseases, including Parkinson's disease (10), Alzheimer's disease (11), and Huntington's disease (12). In glaucoma, immune responses are known to occur at predegenerative stages (5, 8), but key questions remain unanswered (2, 13-15). It is not known whether the earliest immune responses are protective or damaging, or which events irreversibly damage the optic nerve. Moreover, it is not known whether the immune responses are secondary to RGC dysfunction or occur independently, possibly as a more direct result of IOP elevation.In glaucoma, an early insult to RGC axons occurs where they exit the eye in the optic nerve head (ONH) (16)(17)(18)(19)(20). Modeling shows that an increase in IOP inside the eye leads to increased strain in this critical region (21,22). To understand the molecular responses occurring during glaucoma, gene expression profiles of human lamina astrocytes and ONH tissue from animal models hav...

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wlds and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.

ATP8A2 is a P 4 -ATPase that is highly expressed in the retina, brain, spinal cord and testes. In the retina, ATP8A2 is localized in photoreceptors where it uses ATP to transport phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the exoplasmic to the cytoplasmic leaflet of membranes. Although mutations in ATP8A2 have been reported to cause mental retardation in humans and degeneration of spinal motor neurons in mice, the role of ATP8A2 in sensory systems has not been investigated. We have analyzed the retina and cochlea of ATP8A2-deficient mice to determine the role of ATP8A2 in visual and auditory systems. ATP8A2-deficient mice have shortened photoreceptor outer segments, a reduction in photoresponses and decreased photoreceptor viability. The ultrastructure and phagocytosis of the photoreceptor outer segment appeared normal, but the PS and PE compositions were altered and the rhodopsin content was decreased. The auditory brainstem response threshold was significantly higher and degeneration of spiral ganglion cells was apparent. Our studies indicate that ATP8A2 plays a crucial role in photoreceptor and spiral ganglion cell function and survival by maintaining phospholipid composition and contributing to vesicle trafficking.

Autosomal recessive polycystic kidney disease (OMIM 263200) is a serious condition of the kidney and liver caused by mutations in a single gene, PKHD1. This gene encodes Fibrocystin/Polyductin (FPC, PD1), a large protein shown by in vitro studies to undergo Notch-like processing. Its cytoplasmic tail, reported to include a ciliary targeting sequence, a nuclear localization signal and a polycystin-2 binding domain, is thought to traffic to the nucleus after cleavage. We now report a novel mouse line with a triple HA-epitope “knocked-in” to the C-terminus along with lox P sites flanking exon 67, which encodes most of the C-terminus (Pkhd1Flox67HA). The triple HA-epitope has no functional effect as assayed by phenotype and allows in vivo tracking of Fibrocystin. We used the HA tag to identify previously predicted Fibrocystin cleavage products in tissue. In addition, we found that Polycystin-2 fails to co-precipitate with Fibrocystin in kidney samples. Immunofluorescence studies with anti-HA antibodies demonstrate that Fibrocystin is primarily present in a sub-apical location in kidney, biliary duct and pancreatic ducts, partially overlapping with the Golgi. In contrast to previous studies, the endogenous protein in the primary cilia was not detectable in mouse tissues. After Cre-mediated deletion, homozygous Pkhd1Δ67 mice are completely normal. Thus, Pkhd1Flox67HA is a valid model to track Pkhd1-derived products containing the C-terminus. Significantly, exon 67 containing the nuclear localization signal and the polycystin-2 binding domain is not essential for Fibrocystin function in our model.

Defective rhodopsin homeostasis is one of the major causes of retinal degeneration, including the disease Retinitis pigmentosa. To identify cellular factors required for the biosynthesis of rhodopsin, we performed a genome-wide genetic screen in Drosophila for mutants with reduced levels of rhodopsin. We isolated loss-of-function alleles in endoplasmic reticulum membrane protein complex 3 (emc3), emc5, and emc6, each of which exhibited defective phototransduction and photoreceptor cell degeneration. EMC3, EMC5, and EMC6 were essential for rhodopsin synthesis independent of the ER associated degradation (ERAD) pathway, which eliminates misfolded proteins. We generated null mutations for all EMC subunits, and further demonstrated that different EMC subunits play roles in different cellular functions. Conditional knockout of the Emc3 gene in mice led to mislocalization of rhodopsin protein and death of cone and rod photoreceptor cells. These data indicate conserved roles for EMC subunits in maintaining rhodopsin homeostasis and photoreceptor function, and suggest that retinal degeneration may also be caused by defects in early biosynthesis of rhodopsin. Recently, it was shown that EMC functions as a transmembrane domain insertase, acting during the translation process to enable biogenesis of its client transmembrane proteins [13, 14]. EMC functions in multiple cellular processes, including autophagy, lipid transfer, viral infection, Edited by E.

The Drosophila pipe gene encodes ten related proteins that exhibit amino acid sequence similarity to vertebrate heparan sulfate 2-O-sulfotransferase. One of the Pipe isoforms, which is expressed in the ventral follicular epithelium, is a key determinant of embryonic dorsoventral polarity, suggesting that Pipe-mediated sulfation of a heparan sulfate proteoglycan provides a spatial cue for dorsoventral axis formation. We used several approaches to investigate this possibility in the work described here. We determined the nucleotide alterations in 11 different pipealleles. Ten of the mutations specifically affect the pipe isoform that is expressed in the ovary. Among these ten mutations, two alter an amino acid in the putative binding site for 3′-phosphoadenosine 5′-phosphosulfate, the universal sulfate donor. Using Alcian Blue, a histochemical stain that detects sulfated glycans, we observed a novel, pipe-dependent macromolecule in the embryonic salivary glands. Genes known to participate in the formation of heparan sulfate in Drosophila are not required for the production of this material. To investigate whether a heparan sulfate proteoglycan is involved in pipe function in dorsoventral patterning, we generated females carrying follicle cell clones mutant for heparan sulfate synthesis-related genes. Embryos from follicles with mutant clones did not exhibit a dorsalized phenotype. Taken together, our data provide evidence that Pipe acts as a sulfotransferase, but argue against the hypothesis that the target of Pipe is a heparan sulfate glycosaminoglycan.